Mycobacterial species are responsible for several devastating illnesses that affect a huge proportion of the world's population. These Gram positive bacteria infect and replicate within macrophages. They possess a number of characteristics that have impeded advances in knowledge of the basic physiology of the cell. In the face of extremely slow growth rates of the pathogenic species, including M. tuberculosis, and a thick cell wall, recent development of genetic techniques has allowed significant opportunities to study these intractable organisms. The complexity of mycobacterial cell walls renders the bacilli highly impermeable to even small solutes and substrates. Once substrates have penetrated the cell wall, they are transported by permeases across the membrane and into the cell. Our laboratory addresses the problem of membrane transport of nutrients into mycobacterial cells. Using classical and molecular genetic techniques, we first isolate mutants of BCG and M. tuberculosis defective in transport of amino acids and peptides. These nutrients are expected to be major sources of nitrogen, carbon and energy for intracellular bacteria. Peptides may be particularly important since they are the end product of one of the macrophage's primary functions, the breakdown of foreign proteins and organisms. Using radiolabelled substrates and/or an HPLC-based transport analysis system, we then characterize the transport behavior of the mutants, comparing them with wild type. Among the classes of mutants we expect to isolate are those with regulatory mutations. The activity and/or expression of transporters have been demonstrated in other bacterial systems to be regulated by changes in growth conditions. It is our hypothesis that the regulation of nutrient transport in mycobacteria is an important aspect of survival of the organisms in the macrophage. Thus the overall goal of the laboratory is to examine the role of nutrient transport in the survival and replication of M. tuberculosis in primary murine bone marrow- derived macrophages. Using wild type and transport defective mutants of BCG and M. tuberculosis we will characterize the accumulation of amino acids and peptides by intracellular bacteria, by isolating mycobacteria-containing vacuoles from infected cells exposed to radiolabelled substrates. These experiments will lead to a deeper understanding of the intracellular and intravacuolar milieu, of the interaction of mycobacteria with their host cells and of the interaction between the mycobacterial vacuole and the highly dynamic endoplasmic reticulum. Mutant studies will contribute to new antimycobacterial drug design and vaccine development.